The use of a photosynthetic cell extract comprising functional thylakoids in cosmetic compositions

ABSTRACT

The invention relates to cosmetic and topical compositions containing an effective amount of a photosynthetic cell extract comprising a functional thylakoid system. The cosmetic compositions have anti-wrinkle and anti-aging effects on a user&#39;s skin. In addition, the photosynthetic cell extract protects skin against ultraviolet A (UVA) and ultraviolet (UVB) damage.

FIELD OF THE INVENTION

The invention relates to cosmetic and topical compositions containing aneffective amount of a photosynthetic cell extract comprising afunctional thylakoid system. More specifically, it relates to cosmeticcompositions having anti-wrinkle and anti-aging effects on a user'sskin. The invention relates to the use of photosynthetic cell extract toprotect skin against ultraviolet A (UVA) and ultraviolet (UVB) damage.

BACKGROUND OF THE INVENTION

The skin is a complex organ with 3 major tissue layers: the epidermis,dermis and hypodermis. Skin structure, as well as its different celltypes, organization and role have been described in numerouspublications. In order to understand the impact of substances such asdrugs, natural extracts, and ultraviolet radiation on the skin,non-animal tests have been developed and are now used successfully inthe study of skin damage/repair (Auger 2004; Rouabhia 1997; Van de Sandt1999).

Anti-oxidants significantly prevent tissue damage and stimulate woundhealing. This is done through numerous mechanisms includingprevention/limitation of lipid peroxidation, inflammation and alterationof cell DNA. Some plant extracts are believed to have stronganti-oxidant effects (Thang 2001).

Thylakoids are specialized membranes that are responsible forphotosynthesis in eukaryotes (plant and algae) and prokaryotes(bacteria). These photosynthetic organisms convert CO₂ to organicmaterial by reducing this gas to carbohydrates in a complex set ofreactions. Electrons for this reduction reaction ultimately come fromwater, which is then converted to oxygen and protons. Energy for thisprocess is provided by light, which is absorbed by pigments (primarilychlorophylls and carotenoids).

The skin is an interface between the body and the environment and iscontinuously exposed to both endogenous and environmental factors thatcan cause damage and accelerate skin aging. Oxidative stress from freeradicals or reactive oxygen species (ROS) is considered to be a majorcontributor to the process of aging. The ROS are produced by normalchemical reactions in the body as well as by UV radiation, pollution,smoking, stress and other external factors. It has been demonstratedthat, during ageing, ROS levels rise in the skin while the antioxidantdefenses decline. Oxidative stress is involved in the damage of cellularconstituents, such as DNA, cell membrane lipids and proteins. Therefore,antioxidants applied topically can play a key role in reducing thedamage caused by free radicals in the skin.

Lipid peroxidation is a well-established mechanism of cellular injury inboth plants and animals, and is used as an indicator of oxidative stressin cells and tissues. Lipid peroxides, derived from polyunsaturatedfatty acids, are unstable and decompose to form a complex series ofcompounds. These include reactive carbonyl compounds, of which the mostabundant is malondialdehyde (MDA). Measurement of MDA, therefore, iswidely used as an indicator of lipid peroxidation (Esterbaur, 1991).Increased levels of lipid peroxidation products have been associatedwith a variety of chronic diseases in both humans and model systems. Thethiobarbituric acid reactive substances (TBARS) assay is commonly usedto measure MDA in biological samples. However, this reaction isrelatively nonspecific as both free and protein-bound MDA can react.

The MDA-586 method is designed to assay free MDA or, after a hydrolysisstep, total MDA (i.e., free and protein-bound Schiff base conjugates).The assay conditions serve to minimize interference from other lipidperoxidation products, such as 4-hydroxyalkenals.

UVB irradiation (280-320 nm) is well absorbed in various biologicalmacromolecules such as proteins, lipids, and DNA causing damage directlyby converting the irradiation energy to photochemical reactions. Inaddition, ROS (e.g. oxygen radicals and singlet oxygen) are produced,which can modify the cellular DNA and other cellular components,possibly leading to photo-carcinogenesis. The UVA component of solarradiation (320-400 nm) has also been shown to produce deleteriousbiological effects in which singlet oxygen plays a major role. This isof particular importance in tissue that is exposed to UVA irradiation,such as the skin and the eye.

Skin is frequently exposed to sunlight, and UVA exposure is thought tocause skin aging and skin cancer mainly through the action of singletoxygen. Singlet oxygen mediates gene regulation via the transcriptionfactor activator protein-2, activates stress-activated protein kinases,or induces in skin fibroblasts a pattern of mitogen-activated proteinkinase as well as an induction of p38 and c-Jun-N-terminal kinase.

A limited number of molecules in tissue weakly absorb UVA irradiation.After UVA irradiation absorption, these molecules (endogenousphoto-sensitizer) crossover to its long-lived triplet state that allowstransferring energy to oxygen molecules. The transferred energy leads toan energetically excited oxygen molecule (singlet oxygen), which ishighly reactive.

It is well known that t-butyl hydroperoxide (tBHP) mimics the lipidperoxidation on skin (human keratinocytes). tBHP is an organic peroxideused to induce free radical production in several biological systems.Red cells exposed to tBHP undergo lipid peroxidation, haemoglobindegradation and hexose monophosphate-shunt stimulation. Lipidperoxidation and haemoglobin degradation represent extremes of aspectrum of oxidative damage. tBHP induces cell death via apoptosis ornecrosis. Erythrocyte haemolysis assay is one of the best cellularmodels to evaluate the anti-oxidative effect of a compound.

A dynamic and intact thylakoid membrane extract having bothanti-oxidative and anti-inflammatory properties, and its use incombination with other anti-inflammatory compounds, have been describedin International patent publication numbers WO 01/49305 and WO 01/04042,respectively. The anti-oxidative and anti-inflammatory properties of thethylakoid extract have been demonstrated in in vitro, ex vivo, in situand in vivo studies. Specifically, the thylakoid extract has been shownto capture the noxious reactive oxygen species including singlet oxygenspecies, and to modulate pro- and anti-inflammatory cytokines towardattenuation of inflammation.

The use of thylakoid extracts as ROS scavengers, as photoprotectors,particularly against ultraviolet (UV) radiations, and as a solar screenbecause of its capacity to capture UV radiations and to dissipate thesolar energy into heat, has also been described (WO 01/49305).

Furthermore, US 20070036877 discloses that, in vivo, topicalapplications of the thylakoid extract applied directly to the site ofinjury, have been shown to prevent or reduce the UV-induced skin damagein hairless mice.

There is a need for cosmetic and topical compositions containing aneffective amount of a photosynthetic cell extract comprising afunctional thylakoid system (“photosynthetic cell extract” or “extract”)and having anti-wrinkle and anti-aging effects on a user's skin. Thereis also a need for cosmetic and topical compositions containing aneffective amount of the photosynthetic cell extract to provide prolongedprotection of the skin against ultraviolet A (UVA) and ultraviolet (UVB)damage.

SUMMARY OF THE INVENTION

The present invention provides a new use for a photosynthetic cellextract, that is, in a cosmetic composition comprising thephotosynthetic cell extract in anti-aging and anti-oxidant applicationsfor increasing the firmness and hydration of a user's skin and forprotecting the user's skin against UVA and UVB damage.

The invention also relates to the cosmetic treatment of wrinkles bylocal or subcutaneous applications of a cosmetic composition containingthe photosynthetic cell extract.

The invention also relates to the use of a photosynthetic cell extractagainst tissue and DNA damage induced by UVA or UVB radiation, and to acomposition comprising the photosynthetic cell extract and an excipientfor topical administration. The inventors have discovered a surprisingsynergism obtained by combining both the photosynthetic cell extract anda sunscreen to protect skin against UVA and UVB damage.

Furthermore, the inventors have discovered that the addition of aphotosynthetic cell extract to a topical composition will prolong thecomposition's ability to protect the skin from UVA and UVB damage. Acream formulation containing the extract has been shown to protectagainst lipid peroxidation by UV irradiation and to protect againsterythrocyte haemolysis, compared with formulations without the extract.

The photosynthetic cell extract comprises a unique natural antioxidantcomplex that has the ability to continuously capture and dissipatenoxious energy generated by ROS. The extract is, therefore, capable ofcapturing ROS, neutralizing the ROS by dissipating the noxious energygenerated by the ROS and the returning to its original state ready torepeat the cycle over and over again. It is this dynamism and capacityto regenerate that provides the extract with its unprecedented,long-lasting antioxidant protection.

The composition according to the invention can be prepared in andembodied in all pharmaceutical forms normally used for topicalapplication. Furthermore, the composition may comprise the usualadditives in the cosmetic and dermatological fields, such as fats,emulsifiers and co-emulsifiers, hydrophilic or lipophilic gellingagents, hydrophilic or lipophilic active ingredients, preservatives,antioxidants, solvents, fragrances, fillers, hydrophilic and lipophilicfilters, dyestuffs, neutralizers, pro-penetrating agents and polymers.

The extract can be formulated in a liquid composition (a non-lyophilisedextract), a lyophilized extract reconstituted in water, physiologicalsaline or any other solution compatible with topical administration, inpropylene glycol, or in a solid composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the efficacy of the extract against UVB-inducedtissue damage. Damage was observed following exposure to two levels ofUVB exposure (10 and 25 kJ/m²) in untreated tissue (control), tissuetreated with an extract-free composition (vehicle), and tissue treatedwith compositions comprising 0.1% (A) and 0.01% (B) of the extract.

FIG. 2 illustrates the efficacy of the extract against UVA-inducedtissue damage. Damage was observed following exposure to two levels ofUVA exposure (250 and 750 kJ/m²) in untreated tissue (control), tissuetreated with an extract-free composition (vehicle), and tissue treatedwith compositions comprising 0.1% (A) and 0.01% (B) of the extract.

FIG. 3 illustrates the efficacy of the extract against UVB-inducedcyclobutane pyrimidine dimer (CPD) formation.

FIG. 4 illustrates the evaluation of CPD frequency following extracttreatment then UVB irradiation where (1) indicates the damage induced byUVB as judged by the low molecular weight of DNA fragments and (2)indicates the protection offered by the extract, as judged by thepresence of DNA at higher molecular weight than shown in unprotectedtissue.

FIG. 5 illustrates the evaluation of CPD frequency following the extracttreatment then UVB irradiation. Tissue treated with the extract has lessphoto-oxidative damage then the unprotected one as judged by the DNAsmears at low molecular weight.

FIG. 6 illustrates the synergistic effects of the extract and sunscreenagainst UVB-induced tissue damage. Engineered human skins were protectedwith sunscreen (SPF=7.5) alone, sunscreen plus 0.01% extract, orsunscreen plus 0.1% extract for 30 minutes. Control tissues were notprotected. Protected and unprotected tissues were exposed or not to 25kJ/m² of UVB. Immediately after irradiation, biopsies were collected andstained using Masson trichrome. Stained sections were then analyzed andphotographed using an optical microscope at 250× magnification.

FIG. 7 illustrates the synergistic effects of the extract and sunscreenagainst UVA-induced tissue damage. Engineered tissues were protectedwith sunscreen (SPF=7.5) alone, sunscreen plus 0.01% extract, orsunscreen plus 0.1% extract for 30 minutes. Control tissues were notprotected. Protected and unprotected tissues were exposed or not to 750kJ/m² of UVA. Immediately after irradiation, biopsies were collected andstained using Masson trichrome. Stained sections were then analyzed andphotographed using an optical microscope at 250× magnification.

FIG. 8 illustrates the synergistic effects of the extract and sunscreenagainst UVB-induced CPDs. Engineered human skins were protected withsunscreen (SPF=7.5) alone, sunscreen plus 0.01% extract, or sunscreenplus 0.1% extract for 30 minutes. Control tissues were not protected.Protected and unprotected tissues were exposed or not to 25 kJ/m² ofUVB. Immediately after irradiation, biopsies were collected and stainedusing a specific anti-CPD monoclonal antibody. Stained sections werethen analyzed and photographed using a fluorescence microscope at 250×magnification.

FIG. 9 illustrates the synergistic effects of the extract and sunscreenagainst UVB-induced CPDs. Engineered tissues were protected withsunscreen (SPF=7.5) alone, sunscreen plus 0.01% extract, or sunscreenplus 0.1% extract for 30 minutes. Control tissues were not protected.Protected and unprotected tissues were exposed or not to 25 kJ/m² ofUVB. Immediately after irradiation, DNA was extracted from each sample,treated with T4endo-V and fractionated by electrophoresis (C=Control,V=Vehicle, M=Molecular weight standard, SS=Sunscreen).

FIG. 10 illustrates the synergistic effects of the extract and sunscreenagainst UVA-induced photo-oxidative damage. Engineered tissues wereprotected with sunscreen (SPF=7.5) alone, sunscreen plus 0.01% extract,or sunscreen plus 0.1% extract for 30 minutes. Control tissues were notprotected. Protected and unprotected tissues were exposed or not to 750kJ/m² of UVA. Immediately after irradiation, DNA was extracted from eachsample treated with Nth and Fpg then fractionated by electrophoresis(C=Control, V=Vehicle, M=Molecular weight standard, SS=Sunscreen).

FIG. 11 illustrates the efficacy of the extract on repairing UVB-inducedtissue damage. Engineered tissues were exposed to 150 J/m² of UVB.Immediately after irradiation, tissues were or not over layered withvehicle, or the extract at two concentrations: 0.01% and 0.1%. Threehours later, biopsies were collected and stained using Masson trichromeand observed using an optical microscope at 250× magnification.

FIG. 12 illustrates the efficacy of the extract on repairing UVA-inducedtissue damage. Engineered tissues were exposed to 250 kJ/m² of UVA.Immediately after irradiation, tissues were over layered or not withvehicle, or the extract at two concentrations: 0.01% and 0.1%. Threehours later, biopsies were collected and stained using Masson trichomeand observed using an optical microscope at 250× magnification.

FIG. 13 illustrates the efficacy of the extract on repairing UVB-inducedCPD formation. Engineered tissues were exposed to 150 J/m² of UVB.Immediately after irradiation, tissues were over layered or not withvehicle, or the extract at two concentrations: 0.01% and 0.1%. Threehours later, biopsies were collected and stained using anti-CPDmonoclonal antibody and observed using a fluorescence microscope at 250×magnification.

FIG. 14 illustrates the efficacy of the extract on repairing UVB-inducedCPDs. Engineered tissues were exposed to 150 J/m² of UVB. Immediatelyafter irradiation, tissues were over layered or not with vehicle, or theextract at two concentrations: 0.01% and 0.1%. Three hours later, DNAwas extracted from each sample, treated with T4endo-V and fractionatedby electrophoresis (C=Control, V=Vehicle, M=Molecular weight standard).

FIG. 15 illustrates the efficacy of the extract on repairing UVA-inducedphoto-oxidative damage. Engineered tissues were exposed to 250 kJ/m² ofUVA. Immediately after irradiation, tissues were over layered or notwith vehicle, or the extract at two concentrations: 0.01% and 0.1%.Three hours later, DNA was extracted from each sample treated with Nthand Fpg then fractionated by electrophoresis (C=Control, V=Vehicle,M=Molecular weight standard).

FIG. 16 illustrates the percent improvement in hydration at Day 1, Day 7and Day 28 compared to Day 0 as measured by Corneometer®.

FIG. 17 illustrates the evolution of Ue (extensibility) at Day 1, Day 7and Day 28 compared to Day 0.

FIG. 18 illustrates the evolution of Uf (Max. amplitude) at Day 1, Day 7and Day 28 compared to Day 0.

FIG. 19 illustrates the evolution of R9 (fatigability) at Day 1, Day 7and Day 28 compared to Day 0.

FIG. 20 illustrates the evolution of Ur/Ue (firmness) at Day 1, Day 7and Day 28 compared to Day 0.

FIG. 21 illustrates the area of wrinkles at Day 1, Day 7 and Day 28compared to Day 0.

FIG. 22 illustrates the evolution of the total length of wrinkles at Day1, Day 7 and Day 28 compared to Day 0.

FIG. 23 illustrates the evolution of the mean length of the wrinkles atDay 1, Day 7 and Day 28 compared to Day 0.

FIG. 24 illustrates the total number of wrinkles at Day 1, Day 7 and Day28 compared with Day 0.

FIG. 25 illustrates the evolution of the number of wrinkles in Class 1at Day 1, Day 7 and Day 28 compared to Day 0.

FIG. 26 illustrates the evolution of the number of wrinkles in Class 2at Day 1, Day 7 and Day 28 compared to Day 0.

FIG. 27 illustrates the evolution of the number of wrinkles in Class 3at Day 1, Day 7 and Day 28 compared to Day 0.

FIG. 28: illustrates irradiation wells showing fatty acids and creamformulation mixed (1:1) and irradiated for 10 minutes.

FIG. 29 a: illustrates the protection factor of cream formulationsagainst lipid peroxidation caused by UV irradiation (10 minutes); after1 hour incubation (green and blue) and 2 hours of incubation (dark greenand red) at 45° C. (Abbreviations: PBO, placebo cream; PGD2, PurGenesisday (0.01% extract); PGN2, PurGenesis night (0.015% extract); PGE1,PurGenesis eye (0.02% extract); EA, Elisabeth Arden Prevage; LM, LaMer.) Final concentration: fatty acids 10% (v/v); cream formulations:10% (p/v).

FIG. 29 b: illustrates the protection factor of cream formulationsagainst lipid peroxidation caused by UV irradiation (10 minutes); after1 hour incubation (green and blue) and 2 hours of incubation (dark greenand red) at 45° C. The pro-oxidant effect is restricted at 100%.(Abbreviations: PBO, placebo cream; PGD2, PurGenesis day (0.01%extract); PGN2, PurGenesis night (0.015% extract); PGE1, PurGenesis eye(0.02% extract); EA, Elisabeth Arden Prevage; LM, La Mer.) Finalconcentration: fatty acids 10% (v/v); cream formulations 10% (p/v)

FIG. 29 c: illustrates the protection factor of cream formulationsagainst lipid peroxidation caused by UV irradiation (10 minutes); after1 hour incubation (green and blue) and 2 hours of incubation (dark greenand red) at 45° C. (Abbreviations: PBO, placebo cream; PGD2, PurGenesisday (0.01% extract); EA, Elisabeth Arden Prevage; LM, La Mer.) Finalconcentration: fatty acids 10% (v/v); cream formulations 10% (p/v).

FIG. 29 d: illustrates the protection factor of cream formulationsagainst lipid peroxidation caused by UV irradiation (10 minutes); after1 hour incubation (green and blue) and 2 hours of incubation (dark greenand red) at 45° C. The pro-oxidant effect is cut off at 300%. PBO,placebo cream; PGD2, PurGenesis day (0.01% extract); PGN2, PurGenesisnight (0.015% extract); PGE1, PurGenesis eye (0.02% extract); EA,Elisabeth Arden Prevage; LM, La Mer. Final concentration: fatty acids,10% (v/v); cream formulations 10% (p/v).

FIG. 29 e: illustrates the protection factor of cream formulationsagainst lipid peroxidation caused by UV irradiation (10 minutes); after1 hour incubation (green and blue) and 2 hours of incubation (dark greenand red) at 45° C. The pro-oxidant effect is cut off at 200%. PBO,placebo cream; PGD2, PurGenesis day (0.01% extract); PGN2, PurGenesisnight (0.015% extract); PGE1, PurGenesis eye (0.02% extract); EA,Elisabeth Arden Prevage; LM, La Mer. Final concentration: fatty acids,10% (v/v); cream formulations 10% (p/v).

FIG. 30: illustrates the haemolysis of bovine erythrocytes caused bytBHP (2 mM) with and without cosmetic formulations.

FIG. 31: illustrates 150 haemolysis; relative incubation time to provoke50% of cellular damage.

FIG. 32: illustrates the relative protection factor of three cosmeticformulations (0.01%, 0.015% and 0.02% extract concentration).

FIG. 33: illustrates the comparison of the protection factor againstbovine erythrocyte haemolysis for the three cosmetic formulations(0.01%, 0.015% and 0.02% extract concentration), after 125 and 225minutes of incubation.

FIG. 34: illustrates the control (without tBHP): Triplicate plus controlin cream.

DETAILED DESCRIPTION Use of Extract to Protect Skin Against UVA and UVBDamage

In accordance with the present invention, two topical compositions weredeveloped: one comprising 0.01% of the extract and one comprising 0.1%of the extract.

Using artificial sources of UVA and UVB radiation, and topicalcompositions comprising the two extract concentrations, and a topicalcomposition which did not contain any extract, the inventors evaluatedmorphological changes, CPD formation, and DNA damage in engineered humanskin (EHS) when compared with unprotected (control) EHS.

The morphological analysis indicated that the extract providesprotection of EHS against UVA structural damage.

The inventors further discovered that, when added to commercialsunscreen lotion, the photosynthetic cell extract decreasedUVA/UVB-induced DNA damage in the EHS.

The compositions containing the two concentrations of the extractdemonstrated obvious improvements in the repair of EHS structural andDNA damage induced by both UVB and UVA. The inventors discovered,therefore, that the extract promotes the repair of UVA-induced DNAphoto-oxidative damage.

Moreover, it was shown that the addition of a low concentration of theextract (0.01%) to conventional sunscreen demonstrated a surprisinglysignificant increase in the protection against UV induced DNA damage.

Efficacy of the Extract Against UVB-Induced EHS Tissue Damage

The inventors compared EHS treated with topical compositions comprising0.1% extract, 0.01% extract, and no extract (vehicle) with untreated EHS(control). The EHS was exposed to UVB at 10 and 25 kJ/m². As shown inFIG. 1, the different strata (germinativum, granulosum, spinosum andcorneum) of EHS exposed to UVB were less distinguishable from each othercompared to UVB-unexposed tissues. As the UVB dose was increased from 10to 25 kJ/m², there was an increase in epidermal disorganization asdetermined by the thickening of the stratum corneum and reduction in thenumber of epidermal cell layers (FIGS. 1 a and 1 e). Morphologicallydifferentiated keratinocytes (large cells with faint nuclei, largecytoplasm, and the presence of vacuoles) were also induced in theseirradiated tissues. Comparable changes were observed in vehicle-treatedEHS (FIGS. 1 b and 10. Extract-protected EHS showed slight reduction oftissue or cellular damage (FIGS. 1 c, 1 d, 1 g and 1 h). However, thedifferent epidermal layers of both the protected and unprotected EHSremained visible.

These histological analyses suggest that the compositions containing theextract at both concentrations (0.01% and 0.1%) did not act as anefficient tissue structure protector against elevated doses of UVBirradiation (10-25 kJ/m²).

Efficacy of the Extract Against UVA-Induced EHS Tissue Damage

The inventors compared EHS treated with topical compositions comprising0.1% extract, 0.01% extract, and no extract (vehicle) with untreated EHS(control). The EHS was exposed to UVA at 750 and 250 kJ/m². As shown inFIG. 2, the different strata (germinativum, granulosum, spinosum andcorneum) of EHS exposed to UVA were completely disorganized and wereless distinguishable from each other compared to UVA-unexposed tissues.The tissue disorganization, as determined by the thickening of thestratum corneum and reduction in the number of epidermal cell layers(FIGS. 1 a and 1 e), was higher following exposure to 750 kJ/m² comparedto 250 kJ/m². Morphologically, the inventors were unable to identifycells in the UVA irradiated epidermis. Comparable changes were observedin vehicle-treated EHS (FIGS. 1 d and 10 but to a lesser extent.Conversely, extract-protected EHS showed significant reduction of tissueor cellular damage induced by UVA. The different epidermal layers of theprotected EHS remained visible (FIGS. 1 c, 1 d, 1 g, and 1 h) for bothextract concentrations (0.01% and 0.1%). These histological analysesrevealed that the compositions comprising the extract protected tissuestructure against UVA damage.

Efficacy of the Extract Against UVB-Induced EHS DNA Damage.

Using immunofluorescence micrography, the inventors evaluated the effectof the extract on CPD formation and distribution following UVB exposure.As shown in FIG. 3, two doses of UVB, 10 kJ/m² and 25 kJ/m², inducedCPDs in the majority of the epidermal cells in the unprotected (control)EHS (FIGS. 3 b and 3 f). CPD-positive nuclei were distributed throughoutthe full thickness of the epidermis, with a greater proportion ofCPD-stained nuclei in the basal layer. The application of thevehicle-treatment did not prevent the appearance of CPD-positive cells(FIGS. 3 c and 3 g). While the number of CPD-stained cells is slightlyhigher in the unprotected tissue (FIGS. 3 b, 3 f, 3 c and 3 g) comparedto extract-protected (FIGS. 3 d, 3 h, 3 e and 3 i) tissue, theprevention of the formation of CPD was not significant.

The inventors also measured the frequency of CPDs using neutral glyoxalgel electrophoresis. The effects of UVB on the global frequency of CPDsin the epidermis of EHS are shown in FIG. 4, where low molecular weightDNA fragments indicate UVB-induced damage. Analysis of DNA fragmentmobility distribution showed that much smaller DNA fragments werepresent in all treated tissues. After exposure to 10 kJ/m² of UVB, asrevealed by the DNA smears, the compositions comprising the twoconcentrations of extract slightly prevented DNA damage duringirradiation although no protection by the extract was observed at 25kJ/m² irradiation.

These results indicate that the extract at both concentrations (0.01%and 0.1%) did not significantly protect EHS cells against DNA damageinduced by UVB irradiation at 10 and 25 kJ/m².

Efficacy of the Extract Against UVA-Induced EHS Cell Damage.

The inventors also measured the effects of UVA on the frequency of CPDsusing neutral glyoxal gel electrophoresis. The results obtained fromneutral glyoxal gel electrophoresis of DNA digested with Fpg and endoIII (FIG. 5) did not conclusively demonstrate a significant efficacy ofthe extract at either concentration in the protection of EHS cellsagainst DNA damage during UVA irradiation at 250 and 750 kJ/m².

Use of the Extract Plus Sunscreen

The evaluated histological parameters indicate that the addition of theextract to sunscreen (SS) does not reduce the protective effect of thesunscreen against UVA and UVB rays, and does not have a photosensitiveeffect on EHS tissue. But the addition of the extract to a commercialsunscreen demonstrates a surprisingly significant increase in theprotection against UVA and UVB DNA damage when compared to sunscreenalone.

The results demonstrate a synergy between commercial sunscreen and theextract: the addition of the extract to sunscreen significantlyincreases cell DNA protection against UVB induced damage andsignificantly improves its protective capacity against UVA inducedphoto-oxidative damage. Overall, the addition of the extract tosunscreen significantly protected against UVA-induced DNA damage.

Efficacy of the Extract Plus Sunscreen (SS-Extract) Against UVB-InducedEHS Tissue Damage

As shown in FIG. 6, exposure to UVB at 25 kJ/m² induced tissuedisorganization. The different strata (germinativum, granulosum,spinosum and corneum) of EHS exposed to UVB were less distinguishablefrom each other compared to UVB-unexposed tissues. Morphologically,differentiated keratinocytes (large cells with faint nuclei, largecytoplasm, and the presence of vacuoles) present in irradiated tissuesconfirmed the harmful effect of UVB (at 25 kJ/m²). FIGS. 6 f, 6 g and 6h illustrate that the effects of UVB exposure were prevented bysunscreen alone and by the SS-extract mixture. Indeed, the differentepidermal layers of the protected EHS remained visible (FIGS. 6 g and 6h) for both extract concentrations (0.01% and 0.1%) mixed with thesunscreen. In non-irradiated tissue, the SS-extract mixture did notinduce structural changes to the engineered tissues (FIGS. 6 c and 6 d).

Efficacy of SS-Extract Against UVA-Induced EHS Tissue Damage

Results presented in FIG. 7 show that exposure of unprotected tissues to750 kJ/m² of UVA induced tissue disorganization. In irradiated tissues,there was no strata (germinativum, granulosum, spinosum and corneum)differentiation present in unprotected tissue (FIG. 7 a), except thestratum corneum. The stratum corneum was very thick confirming tissueand cell necrosis due to UVA irradiation. The examination of FIGS. 7 gand 7 h reveals that the effects of UVA exposure were prevented bysunscreen alone and by the SS-extract mixture. Indeed, the differentepidermal layers of the protected EHS remained visible (FIGS. 7 f, 7 gand 7 h) for both extract concentrations (0.01% and 0.1%) mixed to thesunscreen. In non-irradiated tissue, the SS-extract mixture did notinduce structural changes to the engineered tissues (FIGS. 7 c and 7 d).

Efficacy of SS-Extract Against UVB-Induced EHS DNA Damage

Using immunofluorescence micrography, the inventors also evaluated theeffect of SS-extract on CPD formation and distribution following UVBexposure. As shown in FIG. 8, exposure to UVB (25 kJ/m²) induced CPDs inthe epidermal cells in the unprotected (control) EHS (FIG. 8 e).CPD-positive nuclei were distributed throughout the full thickness ofthe epidermis, with a greater proportion of CPD-stained nuclei in thebasal layer. The application of sunscreen and SS-extract at bothconcentrations (0.01% and 0.1%) prevented the formation of CPD inirradiated cells. In order to evaluate the synergistic effect betweenthe extract and sunscreen, an assessment using neutral glyoxal gelelectrophoresis was performed. The effects of UVB (25 kJ/m²) on theglobal frequency of CPDs in the epidermis of EHS are shown in FIG. 9.Analysis of DNA fragment mobility distribution demonstrated that muchsmaller DNA fragments were significantly present in unprotected andsunscreen protected EHS. However, when tissues were protected with amixture of SS-extract (0.01% and 0.1%), and exposed to UVB, there wassignificant reduction of the CPD frequency as judged by the presence ofDNA smear at high molecular weight.

Efficacy of SS-Extract Against UVA-Induced EHS DNA Damage

As UVA irradiation is known to produce a significant amount ofphoto-oxidative damage, an assessment using neutral glyoxal gelelectrophoresis on DNA digested with Fpg and endo III was performed.Analysis of DNA fragment mobility distribution showed that much smallerDNA fragments were significantly present in unprotected tissues (FIG.10). In tissue protected with sunscreen alone, the photo-oxidativedamage was prevented as judged by the location of the DNA smear at highmolecular weight. In SS-extract (0.01%) protected tissue the DNA smearwas essentially present at higher molecular weight when compared tounprotected and sunscreen protected tissues.

These results illustrate that the addition of the extract to sunscreenlotion significantly improves its protective capacity against UVAinduced photo-oxidative damage. Overall, the addition of the extract tosunscreen significantly protected against UVA induced DNA damage.

Use of the Extract to Repair UV-Induced Tissue and DNA Damage

Compositions comprising both concentrations of the extract (0.01% and0.1%) demonstrated obvious improvements in the repair of EHS structuraland DNA damage induced by both UVB and UVA. The inventors havedemonstrated that the extract repairs UVB-induced tissue damage, verysignificantly promotes reparation of UVB-induced CPDs and promotes therepair of UVA-induced DNA photo-oxidative damage.

Efficacy of the Extract on Repairing UVB-Induced EHS Tissue Damage.

As shown in FIG. 11, exposure to 150 J/m² of UVB induced tissuedisorganization was not repaired 3 hours following irradiation. Thedifferent strata (germinativum, granulosum, spinosum and corneum) of EHSexposed to UVB were not easily distinguishable from each other andrevealed a complete absence of the basal layer. This suggested thatcells in this irradiated tissue were highly affected by the UVBirradiation and were unable to repair UVB-induced damage. The same canbe said of the vehicle-treated tissues, which revealed differentiatedkeratinocytes (large cells with faint nuclei, large cytoplasm, and thepresence of vacuoles). FIG. 11 demonstrates that all UVB side effectswere repaired in tissues treated with compositions comprising theextract. Indeed, the different epidermal layers of the protected EHSremained visible for both extract concentrations (0.01% and 0.1%).

Efficacy of the Extract on Repairing UVA-Induced EHS Tissue Damage.

FIG. 12 illustrates that exposure to 250 kJ/m² of UVA on unprotected(control) and vehicle-protected tissues induced tissue disorganizationwas not repaired 3 hours later as demonstrated by the absence of thebasal layer and stratum corneum thickening. UVA-induced damage wasrepaired in tissues treated with compositions comprising both extractconcentrations (0.01% and 0.1%), specifically in the basal layer. Thesehistological analyses suggest that the extract promotes the reparationprocess in UVA-damaged tissues.

Efficacy of the Extract on Repairing UVB-Induced EHS DNA Damage.

Using immunofluorescence micrography, the effect of the extract onrepairing UVB-induced CPD was evaluated. As shown in FIG. 13, the numberof CPD-positive cells was very high in unprotected tissue when comparedto the vehicle treated tissues. Conversely, there were no CPD positivecells following the three hours incubation in tissues treated withcompositions comprising both extract concentrations (0.01% and 0.1%)indicating a significantly high (total) repair of UVB-induced CPDs. Theeffect of the extract on the reparation process of CPD was confirmed byneutral glyoxal gel electrophoresis analyses. Indeed, FIG. 14 revealsthat the DNA smear in extract-treated tissues was localized at highmolecular weight when compared to the untreated (control) and vehicletreated tissue.

Efficacy of the Extract on Repairing UVA-Induced EHS Cell Damage.

The reparation efficacy of the extract was also assessed against UVAirradiation using neutral glyoxal gel electrophoresis on DNA digestedwith Fpg and endo III. As shown in FIG. 15, the analysis of DNA fragmentmobility distribution revealed much smaller DNA fragments in untreated(control) and vehicle treated tissues then in extract-treated EHS.Indeed, tissue protected with compositions comprising the extract (0.01%and 0.1%), the DNA smear was essentially present at higher molecularweight.

Cosmetic Efficacy (Hydration, Elasticity and Anti-Profilometry Effects)

In accordance with the present invention, cosmetic compositionscomprising three different concentrations of the extract (0.01%, 0.015%and 0.02%) were developed and compared with commercially availablecosmetic creams. The parameters used were hydration, elasticity andanti-profilometry effects on the skin.

Hydration data, obtained on the forehead, temple, under-eye area, cheeksand chin area of each subject, at Day 1, Day 7 and Day 28 are providedin FIG. 16. Improvements were significant versus Day 0 at all threemeasurement points for Group A and Group B and at Day 28 for Group C.The cosmetic composition of the present invention was associated withthe highest level of statistically significant skin hydration of 17%(versus 15% and 6%) by Day 28.

FIG. 17 illustrates the evolution of Ue, which represents the immediateextensibility or ease of deformation of the skin. A reduction in the Uesignifies improvement in firmness measured by the skin's resistance todeformation. The Ue parameter reduced over time for treatment Group A by5% at Day 1, by 7% at Day 7 and by 26% at Day 28. The reduction at Day 1and Day 7 has significant at p<0.05, while the reduction at Day 28 wassignificant at p<0.01. Treatment Groups B and C also demonstratedreductions in the Ue parameter, notably by 27% for Group B and 26% forGroup C at Day 28. These reductions were statistically significant atp<0.01.

FIG. 18 illustrates the evolution of Uf, which represents the maximalamplitude or maximal deformation of the skin, a parameter that increaseswith age. Treatment Group A demonstrated a significant decrease (atp<0.05) in maximal deformation by −5% at Day 1 versus treatment Groups Band C which did not see an improvement in this elasticity parameter atthe same time of measurement. At Day 7, the maximal deformation of theskin was improved significantly (at p<0.05) in treatment Group A andtreatment Group B (6% vs. 8%) while the improvement noted with thetreatment Group C was not statistically significant. At Day 28 all threetreatment groups demonstrated an improvement in the maximal deformationof the skin as follows: 25% for Group A, 28% for Group B and 27% forGroup C. These improvements were statistically significant at p<0.01.

Skin fatigability is a parameter that generally increases with age. Asseen in FIG. 19, no significant changes due to the treatments wereobserved for Groups A, B and C for skin fatigability at Day 1 and Day 7.However, at Day 28, a decrease in skin fatigability was noted for allthree treatment groups but only the improvement seen in treatment GroupA was statistically significant (at p<0.05).

Ur/Ue represents the net elasticity or firmness that diminishes with ageand is considered to be the most important parameter in the study of theskin's elasticity. At Days 1 and 7, as shown in FIG. 20, no significantchanges resulting from the treatments were observed for this parameter.However, treatment with the claimed cosmetic composition demonstrated astatistically significant increase of 5% in firmness by Day 28 (p<0.05).

As shown in FIGS. 21, 22 and 23, the cosmetic composition of the presentinvention showed statistically significant improvements in the followingwrinkle parameters compared to baseline as early as Day 1 of treatmentwhich increased in magnitude with treatment and remained significant atDay 28: area of wrinkles −12% at Day 1 and -17% at Day 28 (p<0.01, each)(FIG. 21); total length of wrinkles −11% at Day 1 and −13% at Day 28(p<0.01, each) (FIG. 22); and mean length of wrinkles −5% at Day 1 and−11% at Day 28 (p<0.05 and p<0.01) (FIG. 23). Significant improvement of−7% (p<0.01) in the total number of wrinkles at Day 1 was also observedwith the cosmetic composition of the present invention as seen in FIG.24. In contrast, treatment with the other two prestige product linesimproved wrinkle parameters but the improvements were only 18 to 47% ofthe magnitude of the improvements seen for the cosmetic composition ofthe present invention and were not statistically significant compared tobaseline.

With respect to the class of the wrinkles, and as shown in FIGS. 25, 26and 27, the cosmetic composition of the present inventionnon-significantly reduced the number of Class 3 (profound) wrinkles by−7% as early as Day 1 and was the only treatment that produced asignificant −13% (p<0.05) reduction from baseline in Class 3 wrinkles atDay 28 of treatment and a −9% (p<0.01) significant reduction in Class 2(moderate) wrinkles at Day 1 of treatment. The other two comparativeprestige product lines increased Class 3 wrinkles at Day 28 by +7% and+5%, respectively.

No significant results were seen in the reductions of the number ofwrinkles of Class 1 (fine) for any of the three treatments. Statisticalanalysis of the depth of the wrinkles revealed no significant reductionover time for any of the three treatments.

Synergistic Effect with Spilanthol

Spilanthol is an Acmella oleracea extract that is known to inhibitcontractions in subcutaneous muscles and to be used as an anti-wrinkleproduct. The use of the spilanthol with the photosynthetic cell extractcauses a synergistic effect in an anti-wrinkle cosmetic composition.

Example 1 Preparation of Engineered Human Skin

Skin donors were healthy women, 15 to 20 years of age. Keratinocytes andfibroblasts were isolated from UV-unexposed normal human skin biopsiesfollowing breast reductive surgeries as previously described. Engineeredhuman skins (EHS) were produced by mixing calf skin type I and type Illcollagen (2 mg/ml, Sigma) with normal human fibroblasts (1.5×10⁶cells/ml) to produce the dermis. Tissues were cultured in 5% fetal calfserum-supplemented medium for 4 days and then seeded with keratinocytes(9×10⁴/cm²) to obtain EHS. EHS were grown under submerged conditions forseven days and were then raised to an air-liquid interface for five moredays to allow the differentiation of the epidermis into the differentstrata. Each series was conducted using keratinocytes and fibroblastsisolated from the same skin biopsy.

Extract Treatment and UV Irradiation.

Two concentrations of the extract (A=0.1% and B=0.01%) were tested. Theextract vehicle alone (at the same concentration found in extracttreatments) served as the first control. Normal untreated tissue servedas the second control. Volumes of 60 □l of the extract or its vehiclewere applied on the stratum corneum of EHS 30 min before irradiation.Three experimental conditions (untreated, vehicle-treated orextract-treated) were tested. Prior to irradiation, the culture mediumwas replaced by the irradiation medium (DME supplemented with bovinepituitary extract), without phenol red and hydrocortisone, in order toavoid UV-induced formation of medium-derived toxic substances. Petridishes containing EHS were placed on ice and uncovered to allow directexposure of EHS to UV rays. Three doses of UVA (0, 250 and 750 kJ/m²)and three doses of UVB (0, 10 and 25 kJ/m²) were used to irradiatetreated and untreated EHS. The UVA source was a neon BLB light 45 cm(number BL-18, 15 W UV, Ateliers Albert Inc., Montreal, QC) with anemission spectrum containing a peak at approximately 360 nm. The UVBsource was a FS20T12/UVB/BP lamp (Philips, Somerset, N.J.) with anemission spectrum containing a peak between 290 and 320 nm. Alladministrated doses were monitored using a YSI Kettering 65A radiometer(Yellow Springe Instruments, Ohio).

Histological and Immunohistochemical Analyses Following UV Exposure.

Immediately after irradiation, biopsies were taken from each EHS. Theywere either fixed with Bouin's solution and then embedded in paraffin,or directly embedded at optimal cutting temperatures, frozen in liquidnitrogen, and stored at −80° C. until use. Thin sections (4 □m) of theparaffin embedded biopsies were stained with Masson Trichrome toevaluate the structure of the tissue as described elsewhere. For CPDevaluation, only tissues irradiated with UVB were used. For thispurpose, thin cryostat sections (4 □m) of UVB-irradiated frozen biopsieswere incubated for 45 min at room temperature with specific mousemonoclonal CPD antibody (Biomedical Technologies, Stoughton, Calif.).The CPD antibody reacts specifically with UV-induced thymidine dimers indouble or single-stranded DNA. Sections were then incubated influorescein isothiocyanate-conjugated (FITC) to goat anti-mouseimmunoglobulin (Chemicon, Temecula, Calif.), diluted 1:100, for 30 minat room temperature. The sections were extensively washed with phosphatebuffered saline between incubations. They were mounted with coverslipsin 50% glycerol mounting medium and observed using epifluorescencemicroscopy and photographed.

Molecular Analyses Following Solar UV Radiation Exposure.

Immediately after irradiation, epidermal cells were isolated aspreviously described. After homogenization, cells were centrifuged andcellular pellets were re-suspended in 2 ml of 0.15 M NaCl; 0.005 M EDTApH 7.8 and 2 ml of 0.02 M Tris-HCl pH 8.0; 0.02 M NaCl; 0.02 M EDTA pH7.8; 1% SDS. DNA was purified as previously described, and used toevaluate the global frequency of CPD photoproducts that are specific toUVB irradiation, and photo-oxidative damage that is specific to UVAirradiation.

To specifically cleave CPDs, 10 μg of UV-irradiated DNA was dissolved in50 μL H₂O. The following solution was added to each DNA sample: 50 μL ofa solution containing 10 μL of 10× dual buffer (10× dual buffer: 500 mMTris-HCl pH 7.6, 500 mM NaCl, and 10 mM EDTA), 0.1 μL of 1 M DTT, 2 μLof 5 mg/mL BSA, a saturating amount of T₄ endonuclease V, and completedwith H₂O to a final volume of 50 μL. The reaction was performed at 37°C. for 1 h. To specifically cleave photo-oxidative damage, 10 μg ofUV-irradiated DNA was dissolved in 50 μl of water and 50 μl of 2× Nthprotein buffer (100 mM tris-HCl pH 7.6, 200 mM KCl, 2 mM EDTA, 0.2 mMdithiothreitol, 200 μg/ml bovine serum albumin). Enzymes (nth and fpg)were added to 5 μl of dilution buffer (50 mM tris-HCl pH 7.6, 100 mMKCl, 1 mM EDTA, 0.1 mM dithiothreitol, 500 μg/ml bovine serum albumin,10% (v/v) glycerol), the total digestion volume was 105 μl. The sampleswere incubated at 37° C. for 60 min. Following ethanol precipitation,digested DNA was re-suspended to a final concentration of 1 μg/μL.

The global frequency for each class of photoproducts was determined withneutral agarose gel electrophoresis ofglyoxal/dimethylsulfoxide-denatured genomic DNA as previously described.Briefly, 5 μg/10 μl of treated DNA was dissolved in distilled water and2 μL of 100 mM sodium phosphate, pH 7.0, 3.5 μL of 6 M glyoxal (SigmaChemical Co., St. Louis, Mo.), and 10 μL of dimethylsulfoxide was added.DNA samples were incubated at 50° C. for 1 h. Prior to loading, 3.8 μLof loading buffer (10 mM sodium phosphate, pH 7.0; 50% glycerol; 0.25%xylene cyanol FF) were added. The gels were run in 10 mM sodiumphosphate pH 7.0, running buffer at 3-4 volts/cm with constant buffercirculation. The gels were stained for 2 h in a solution of 1×SYBR® Goldnucleic acid gel stain (S-11494) (Molecular Probes, Eugene, Oreg.) inTAE pH 8.0 and photographed. The overall adduct frequency was estimatedfollowing the enzymatic conversion of DNA photoproducts to single-strandbreaks. The migration of the DNA fragments through the agarose gelallows for their separation according to their molecular weight—thesmaller the fragment, the greater the distance of migration. Willis etal. have shown that when a randomly cleaved DNA molecule isgel-fractionated, the mobility of each fragment is proportional to thelog of the molecular weight throughout the middle of the mobility range.It is, therefore, possible to calculate the approximate mass of each DNAsmear by estimating the molecular weight at the highest intensity of theDNA staining dye. The numbers obtained were divided by 2 (as eachfragment contains one photoproduct at each end) and expressed as numberof lesions per megabase (Mb).

Example 2 Preparation of Engineered Human Skin

Skin donors were healthy women, 15 to 20 years of age. Keratinocytes andfibroblasts were isolated from UV-unexposed normal human skin biopsiesfollowing breast reductive surgeries as previously described. Engineeredhuman skins were produced as described above.

Sunscreen Plus Extract Treatment and UV Irradiation.

Two concentrations of the extract (0.01% and 0.1%) were mixed v/vseparately with SPF 15 sunscreen (SS). After mixing with the extract(0.01% and 0.1%), the obtained sunscreen had a SPF of 7.5. The vehicle(sunscreen with a SPF 7.5) served as a control. Normal unprotectedtissue served as a second control. Volumes of 60 μl of SS-extract orSS-vehicle were applied on the stratum corneum of EHS for 30 minutesbefore irradiation. The irradiation procedures were the same asdescribed above. Two doses of UVA (0 and 750 kJ/m²) and two doses of UVB(0 and 25 kJ/m²) were used to irradiate protected and unprotected EHS.

Histological and Immunohistochemical Analyses Following UV Exposure.

Immediately after irradiation, biopsies were taken from each EHS. Theywere either fixed with Bouin's solution and embedded in paraffin, ordirectly embedded at optimal cutting temperature, frozen in liquidnitrogen, and stored at −80° C. until use. Histological (Massontrichrome staining) and immunofluorescence (CPDs) analyses wereperformed as described above. For CPD evaluation, only tissue irradiatedwith UVB was used.

Molecular Analyses Following UV Exposure.

Immediately after irradiation, epidermal cells were isolated and used toextract DNA. Purified DNA was used to evaluate the global frequency ofCPD photoproducts that are specific to UVB irradiation, andphoto-oxidative damage that is specific to UVA irradiation. For thispurpose, the inventors used the different steps described above.

Example 3 Preparation of Engineered Human Skin

Skin donors were healthy women, 15 to 20 years of age. Keratinocytes andfibroblasts were isolated from UV-unexposed normal human skin biopsiesfollowing breast reductive surgeries as previously described. Engineeredhuman skins were produced as described above.

Tissues Exposure to UV Followed by Treatment with the Extract.

After their production, EHS was exposed to ultraviolet (UVA or UVB)sources. One dose (250 kJ/m²) of UVA and one dose (150 J/m²) of UVB wereused to irradiate unprotected EHS. The irradiation procedures were thesame as described in section 1.2. Immediately after irradiation tissueswere treated with the extract. Volumes of 60 μl of the extract or itsvehicle were applied on the stratum corneum of EHS for 3 hours prior toanalysis.

Histological and Immunohistochemical Analyses.

Following the incubation period, biopsies were taken from each EHS. Theywere either fixed with Bouin's solution and embedded in paraffin, ordirectly embedded at optimal cutting temperature, frozen in liquidnitrogen, and stored at −80° C. until use. Histological (Masson trichomestaining) and immunofluorescence (CPDs) analyses were performed asdescribed above. For CPD evaluation, only tissues irradiated with UVBwere used.

Molecular Analyses Following UV Exposure.

Following the incubation period, epidermal cells were isolated and usedto extract DNA. Purified DNA was used to evaluate the global frequencyof CPD photoproducts that are specific to UVB irradiation, andphoto-oxidative damage that is specific to UVA irradiation.

Example 4

In a comparative cosmetic efficacy study (single-blind, mono-centric,parallel group design) of 72 healthy female volunteers aged 35 to 72years (mean age 54.6 years), the efficacy of the present cosmeticcomposition was compared to that of two leading commercial anti-agingbrands over a 28 day period of use. Efficacy parameters included: effecton skin appearance, hydration, elasticity, and profilometry(anti-wrinkle effect).

Each volunteer was provided with three formulations of the presentcosmetic composition comprising 0.01%, 0.015% and 0.02% of the extract,in combination with non-active ingredients, along with applicationinstructions, to be used over a 28-day period. Measurements were takenon Day 0, Day 1, Day 7 and Day 28. Hydration was assessed using aComeometer®, elasticity was assessed using a Cutometer® and profilometrymeasurements were taken from silicone replicates of the eye contourzones using a Visia-CR Imaging system. Verification of product usage wasdetermined by weighing the product samples.

On the first visit (Day 0 or D0) the volunteers randomly received thecontainers of one of the three test treatments (day cream, eye lotionand night cream), a follow-up sheet to be completed after everyapplication and a self-evaluation questionnaire to be completed after 28days of treatment.

The volunteers were instructed to apply, each morning, after havingwashed their face and hands (with their regular cleansing products) asufficient quantity of the eye lotion to cover the eye contour areaincluding the crows' feet area of their face. After the eye lotion waswell penetrated, the volunteers had to apply the day cream, insufficient quantities, to cover their entire face avoiding the eyecontour area.

Additionally, the volunteers were instructed to apply, every evening,enough of the night cream to cover their entire face avoiding the eyecontour area.

The use of all other skin care products (except for regular cleansingproducts and makeup) was prohibited during the study. Changes regardingthe brand of their regular facial cleanser or makeup products were notpermitted during the week prior the commencement of the study nor duringthe study.

On Day 0, twelve digital photographs of the face (full front, rightprofile and left profile, in four different imaging modes: Standard,Cross Polarized, UV. and Parallel Polarized), were taken using theVisia-CR Imaging System. Subsequently, the study was conducted in alaboratory room with controlled temperature (22° C.±3) and relativehumidity (30%±5). After 15 minutes of stabilisation in the controlledroom, measurements of hydration using Corneometer®, measurements ofelasticity using Cutometer®, and Profilometry by silicone imprints ofeye contour zones before treatment were taken.

Measurements taken at D0 were repeated at D1, D7 and D28 in the samemanner as described above. At D1, D7 and D28 the volunteers had toreturn their completed daily logs. They were also to return to the tabthe sample containers with the unused portion of the test products. Theunused portion of the sample containers and daily logs (use diaries)were intended for verification of the volunteers' adherence to theprotocol.

Hydration.

Epidermal moisture of the stratum corneum can be assessed bynon-invasive in vivo instrumental testing methods based on the electricproperties of the skin, the electrical capacitance. The stratum corneumis a dielectric corpus and all changes in its hydration status arereflected by changes in the electric capacitance, expressed in arbitraryunits by the Comeometer®.

Elasticity.

The skin's appearance is related to and highly affected by its elasticproperties. The elasticity of the skin is subject to change with the useof cosmetic products. Changes in the mechanic and viscoelasticproperties of the skin reflect the elasticity of the skin. Theelasticity related parameters were measured by Cutometer® SEM 575(Courage and Khazaka, Germany). The instrument is equipped with a probe(2 mm aperture in diameter) that includes a controlled suction (vacuumof 400 mbar) on the skin with four repetitions of 1 second. Twomeasurements were taken from the middle of each cheek.

The results on each measurement site are expressed as the followingparameters:

-   -   Ue=“extensibility” or “immediate elastic deformation” due to the        application of vacuum    -   Uf=“total amplitude” or “maximum amplitude” of the skin (Ue+Uv)    -   Ur=“tonicity”    -   Ur/Ue=“net elasticity” or “pure elasticity” or firming

Finally, after four aspirations, the Cutometer® provides a measure ofthe skin's “fatigability”.

Anti-Wrinkle Effect.

Imprints (negatives of the skin surface) of the eye contour zones wereobtained by applying silicone polymer onto the “crows' feet” area of theeye contour zone, while the volunteer maintained an upright but sittingposition. The silicone polymer used for this study consisted of Silflo®(silicone dental impression material of Flexico-Developments Ltd.,Potters Bar, England).

Imprints of the crows' feet were analyzed by a computerized digitalimage processing system coupled to Quantirides® software (designed byMonoderm, Monaco) to obtain the topography of the skin. This standardtechnique is based on measuring the shadows cast when an incident lightis inclined at an angle of 35° on the replica.

The analyzed parameters were the total area of wrinkled skin, the numberand the mean depth of the depressions due to the cutaneous relief, anddepth of deep and medium wrinkles. The wrinkles were differentiated bydepth (Class 1 for 0-55 μm; Class 2 for 55-100 μm; and Class 3 for110-800 μm) before and after treatment in order to better demonstratethe efficacy of a given product.

What is claimed:
 1. A cosmetic composition comprising: an effectiveamount of a photosynthetic cell extract, wherein the extract comprisesfunctional thylakoids, and an acceptable carrier, and wherein thecomposition optionally further comprises sunscreen.
 2. The compositionof claim 1, wherein the extract is present in an amount from about 0.01%to about 0.1% based upon the total weight of the composition.
 3. Thecomposition of claim 1, formulated for topical application.
 4. Thecomposition of claim 1, formulated for subcutaneous application
 5. Thecomposition of claim 1, wherein the carrier is selected from the groupconsisting of fats, emulsifiers, co-emulsifiers, hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic active ingredients,preservatives, antioxidants, solvents, fragrances, fillers, hydrophilicand lipophilic filters, dyestuffs, neutralizers, pro-penetrating agentsand polymers.
 6. The composition of claim 1, wherein the extract isnon-lyophilized and formulated in a liquid composition.
 7. Thecomposition of claim 1, wherein the extract is a lyophilized extractreconstituted in water, in physiological saline or any other solutioncompatible with topical administration, in propylene glycol, or in asolid composition.
 8. Use of an effective amount of the compositionaccording to claim 1, for improvement of the aesthetic appearance ofskin, said improvement selected from the group consisting of: decreasingthe number and depth of wrinkles in the skin, increasing elasticity ofthe skin, increasing hydration of the skin, and a combination thereof.9. Use of an effective amount of the composition according to claim 1,to provide prolonged protection to skin against oxidative damage causedby ultra-violet (UV) radiation.
 10. Use of an effective amount of thecomposition according to claim 1, to treat skin damaged by ultra-violet(UV) radiation.
 11. (canceled)
 12. (canceled)
 13. The use of claim 9,wherein the oxidative damage is lipid peroxidation.
 14. The use of claim9, wherein the oxidative damage is erythrocyte haemolysis.
 15. The useof claim 9, wherein the oxidative damage is DNA damage.
 16. Use of aphotosynthetic cell extract for the preparation of a cosmeticcomposition for improvement of the aesthetic appearance of skin, saidimprovement selected from the group consisting of: decreasing the numberand depth of wrinkles in the skin, increasing elasticity of the skin,increasing hydration of the skin, and a combination thereof, wherein theextract comprises functional thylakoids.
 17. Use of a photosyntheticcell extract for the preparation of a cosmetic composition to provideprolonged protection to skin against oxidative damage induced byultra-violet (UV) radiation, wherein the extract comprises functionalthylakoids, and wherein the composition comprises sunscreen.
 18. Use ofa photosynthetic cell extract for the preparation of a cosmeticcomposition to treat skin damaged by ultra-violet (UV) radiation,wherein the extract comprises functional thylakoids.
 19. (canceled) 20.(canceled)
 21. The use of claim 17, wherein the oxidative damage islipid peroxidation.
 22. The use of claim 17, wherein the oxidativedamage is erythrocyte haemolysis.